Abstract Body: Introduction: Type 1 Diabetes (T1D) is an autoimmune disease that affects nearly 2 million people in the U.S. Extracellular Vesicles (EVs) are small vesicles released from cells that contain various molecular components, including proteins, lipids, and RNA. They are involved in intercellular communication, influencing various biological processes. In the context of T1D, beta cells (β-cell) under stress may release EVs that contain autoantigens and RNA transcripts that could contribute to the pathogenesis of T1D.
Hypothesis: Test whether β-cell EVs communicate inflammatory signaling to primary macrophages (Mθ) and Cd4+ T cells.
Methods: EndoC-βH1 cells were cultured on Matrigel/fibronectin-coated culture flasks to 75% confluency and subsequently cultured for 48 hours in control serum-free medium (CON-EVs) or medium supplemented with IL-1β, Tnf-α and interferon-gamma (STRESS-EVs) for 48 hours. EVs were purified from the conditioned cell culture medium using ultracentrifugation. CON-EVs and STRESS-EVs were assessed for their ability to communicate immunometabolism signaling to BMDMs and splenic CD4+ T cells via sequencing. Glycolytic metabolism and oxidative phosphorylation (OxPHOS) in both cell types were performed by Seahorse. Mitochondrial membrane potential (TMRM) and mass were detected by flow cytometry.
Results: Bioenergetic assessment showed that while both types of β-cell EVs drove a similar upregulation of glycolytic metabolism, only CON-EVs drove a robust upregulation of OxPHOS, demonstrating that STRESS-EVs shift both Mθ and T cell metabolism to a more effector phenotype by inducing glycolytic dependency. Mitochondrial assays revealed a reduction in TMRM and an accumulation in reactive oxygen species, suggesting the potential detrimental effects of STRESS-EVs. Small RNA sequencing revealed that STRESS-EVs induced an increase in subsets of miRNAs implicated in NFkb-driven inflammation, insulin response and T1D pathogenesis. β-cell EVs modulated a plethora of transcriptional genes across the immune landscape of the recipient immune cells including genes involved in lipid biosynthesis, the autoimmune response and tissue-damaging phenotypes that have been identified as precursors to T1D development. Lastly,STRESS-EVs inhibited Mθ efferocytosis, demonstrating their ability to mediate Mθ functionality.
Conclusion: β-cell EVs modulate MicroRNA-controlled immunometabolism of Mθ and T cells suggesting their capacity to contribute to the pathogenesis of T1D.